The present invention relates to the field of biotechnology. More particularly, it concerns methods and constructs for producing retroviral particles in vitro, ex vivo or in vivo. It also concerns the use of such methods and constructs for transferring nucleic acids into cells.
Retroviruses are currently one of the main types of vector for transferring nucleic acids into cells. They are used for example for transferring nucleic acids in vitro or ex vivo (experimental studies, study of regulation, production of recombinant proteins, introduction of resistance genes or toxicity genes) or directly in vivo (establishment of animal pathological models, labeling or bioavaiiability studies, therapeutic use by grafting producer cells or injecting purified virus, etc.).
For all these utilizations, it is therefore important to have effective methods available for producing and administering retroviruses. In this respect, conventional methods are based on the use of packaging cell lines. Such cell lines are constructed in vitro and express the whole set of proteins required for constituting and packaging a defective retroviral vector in a retroviral particle. Such cell lines are exemplified by the cell lines PSICRIP [Danos and Mulligan, PNAS (1988) 85: 6460], PA317 [Miller and Buttimore, Mol. Cell. Biol. (1986) 6: 2895], or GP+ Env AM12 [Markowitz et al., Virology (1988) 167: 400]. However, the use of such cell lines can pose certain problems, related first to their construction and second, to their use.
For example, such cell lines must be stable, that is, express the retroviral functions in a continuous manner, without genetic rearrangements or loss of expression. In addition, such cell lines must be compatible with the potential pharmacological use of the retroviruses, and must therefore be established from cells that can be cultured, having little or no immunogenicity, etc.
Furthermore, it is important that the cell lines used produce fairly high viral titers free of replication-competent virus (RCV). The methods of retroviral preparation by means of packaging cell lines therefore necessarily comprise quality controls on both the viral stocks produced and the cell lines used. Finally, depending on the desired application, these production methods may have to be carried out under a high level of confinement, further complicating the industrial scalability of such methods.
To address these drawbacks, patent application WO 95/22617 proposed a new concept for producing retroviral particles that avoids the use of retrovirus packaging cells. This new concept consists in transforming a target cell in vitro, ex vivo or directly in vivo to a retrovirus producing cell, by introducing into this cell the whole set of genetic elements required for constituting a retroviral particle. This application provides in particular for transferring these elements by means of plasmid constructs or viral vectors of different origin. As a matter of fact, Feng et al. [Nature Biotechnology (1997) 15: 866] have described a specific embodiment of this new approach, using two first generation recombinant adenoviruses (i.e. defective for the E1 region) into which the retroviral genetic elements have been distributed in a precise arrangement.
The present application now describes a further improvement to the retroviral particle production methods described hereinabove, and the use for transferring nucleic acids.
The present application is based in particular on the use of recombinant adenoviruses for delivering to cells, in vitro, ex vivo or in vivo, the whole set of genetic elements required for constituting a retroviral particle. As compared to previously described works, the present application is based in part on the use of specific types of recombinant adenovirus and/or on a specific distribution of the retroviral genetic elements. As demonstrated in the present application, the methods and constructs according to the invention make it possible to obtain high levels of retroviral particle production, are effective, and display increased safety in vitro, ex vivo as well as in vivo. Furthermore, the defective recombinant retroviruses so produced are infectious, and are capable of transferring a nucleic acid into a cell with high efficiency.
A first subject of the invention therefore concerns a composition comprising the whole set of genetic elements required for constituting a retroviral particle, incorporated in one or several recombinant adenoviruses defective for all or part of the E1 and E4 regions at least.
In the context of the invention, the term xe2x80x9cgenetic elements required for constituting a retroviral particlexe2x80x9d refers to the whole set of nucleic acid sequences, coding and regulatory, which in cis or in trans are necessary and sufficient to constitute a retroviral particle, that is, a physical particle expressing the retroviral envelope at its surface, and inside of which are found the different proteins required to carry out a retroviral replication cycle, and a genome in a form which allows it to be reverse transcribed.
The organization of the retroviral genome is well understood, and comprises primarily the following elements:
An LTR region located at each end of the retroviral genome, serving in particular as origin of transcription and transcriptional promoter. This LTR region more specifically contains elements designated U3, R and U5 (see FIG. 5, for example). The U5 sequence, together with the U3 sequence, plays a key role during provirus integration.
A packaging sequence (Psi or "psgr"), involved in packaging the retroviral genome in the viral particle. The packaging sequence may further contain a region extending to certain elements of the gag gene, which have been reported to improve packaging efficiency.
Three coding regions, named gag, pol and env, coding for the core proteins (gag), enzymes (reverse transcriptase, protease, integrase), and the retroviral envelope (env), respectively.
To constitute a recombinant retroviral particle, a retroviral vector is generally constructed comprising genetic elements acting in cis, i.e., the LTR regions and the packaging sequence, and in which all or part of the gag, pol and/or env coding regions (acting in trans) have been deleted. Normally, such a retroviral vector also comprises a nucleic acid of interest (transgene). The coding regions not present or inactive in the retroviral vector are provided in trans (complementation functions) so as to be able to reconstitute a retroviral particle.
In a specific embodiment of the invention, the genetic elements therefore comprise a retroviral vector and nucleic acids coding for retroviral complementation functions (that is, for functions defective in the retroviral vector, eg., gag, pol and/or env).
In a more particular embodiment of the present invention, the genetic elements comprise:
a nucleic acid coding for a retroviral gag protein,
a nucleic acid coding for a retroviral pol protein,
a nucleic acid coding for an envelope protein, for example a retroviral env protein, and
a nucleic acid comprising, between two LTR regions, a retroviral packaging sequence and a nucleic acid sequence of interest.
To carry out the present invention, the genetic elements may be derived from different types of retrovirus, such as ecotropic and/or amphotropic viruses. In particular, these may be retroviruses belonging to the family of oncoviruses, lentiviruses or spumaviruses. In the oncovirus family, particular examples include the slow oncoviruses, which do not carry an oncogene, such as for example MoMLV, ALV, BLV or MMTV, and rapid oncoviruses, such as RSV for example. In the lentivirus family, examples include HIV, SIV, FIV or CAEV.
Furthermore, the LTR region or regions used may either be complete retroviral LTR regions, or subdomains allowing reconstitution of complete LTR regions after reverse transcription. Thus, the LTR regions used may comprise deletions of domains such as U3 and U5 for example. In a specific embodiment of the invention, the retroviral vector comprises a 5xe2x80x2 LTR deleted for the U3 region and a 3xe2x80x2 LTR deleted for the U5 region. The use of this type of construct allows in particular to stabilize the vector during the construction steps and to lower the risks of recombination. The genetic elements may be directly prepared from isolated retroviruses, according to methods known to those skilled in the art, and/or synthesized artificially. Such elements may be amplified in culture, as described in the examples. Moreover, the nucleic acids used may be DNA or RNA.
In a preferred variant, the genetic elements are such that they code for the retroviral gag and pol proteins and for an envelope protein allowing infection of human cells. More preferably still, the gag and pol proteins are proteins from retroviruses chosen from among MoMLV, ALV, BLV, MMTV or RSV, and the envelope protein is a protein from viruses chosen from A4070, GALV, RD114, VSV-G or rabies virus. It is understood that the invention may also be carried out by using variants or mutants of these proteins, having conserved their biological activity, or chimeric or hybrid proteins allowing to modify the tropism of the retroviral particle, especially by giving it specificity for certain cell types.
In a particular embodiment, the envelope protein is therefore a viral or cellular protein, allowing the retroviral particles to infect human cells. It is preferably a protein from a virus such as A4070, GALV, RD114, VSV-G or rabies virus. The preferred viral envelopes are A4070, GALV or VSV-G. The preferred gag and pol proteins come from the MoMLV retrovirus.
According to a specific embodiment of the invention, the genetic elements allow constitution of a lentivirus particle, and therefore derive, at least in part, from a lentivirus, such as HIV, SIV, FIV or CAEV in particular. The use of recombinant retroviruses prepared from lentiviruses has been described in the literature and offers certain advantages for transferring genes into quiescent cells [Poeschia et al., Nature Medicine (1998) 4: 354]. The present invention now makes it possible to exploit this property to achieve, under improved conditions, gene transfer by lentiviruses.
The disposition of the retroviral genetic elements in the adenovirus or adenoviruses may be accomplished in several ways.
Thus, in one variant embodiment, the invention concerns a composition comprising the whole set of genetic elements required for constituting a retroviral particle distributed into three separate recombinant adenoviruses.
In this particular embodiment, a composition according to the invention comprises for example a first recombinant adenovirus comprising, incorporated in its genome, the nucleic acid or acids encoding gag and pol; a second adenovirus comprising, incorporated in its genome, a nucleic acid encoding the envelope protein; and a third recombinant adenovirus comprising, incorporated in its genome, the retroviral vector.
In another, preferred variant embodiment, the invention concerns a composition comprising the whole set of genetic elements required for constituting a retroviral particle distributed into two separate recombinant adenoviruses. In this respect, an especially preferred composition in the context of the invention comprises:
a first recombinant adenovirus comprising, incorporated in its genome, one or several nucleic acids coding for the retroviral proteins gag and pol, and
a second recombinant adenovirus comprising, incorporated in its genome, a nucleic acid coding for an envelope protein such as defined hereinabove, and a nucleic acid comprising, between two complete LTR regions or subdomains allowing reconstitution of complete LTRs following reverse transcription, a retroviral packaging sequence and a nucleic acid sequence of interest.
The use of such a composition is illustrated in a general way in FIG. 1. As shown in the examples, this specific distribution of retroviral genetic elements makes it possible to obtain high titers of infectious retroviral particles, capable of efficiently and stably transferring a transgene into the cells of interest.
In this respect, a specific subject of the present application concerns
any defective recombinant adenovirus, characterized in that it comprises, incorporated in its genome, one or several nucleic acids coding for the retroviral proteins gag and pol, and
any defective recombinant adenovirus, characterized in that it comprises, incorporated in its genome, a nucleic acid coding for an envelope protein, and a nucleic acid comprising, between two LTR regions complete or not, a retroviral packaging sequence and a nucleic acid sequence of interest.
In particular, such adenoviruses may be defective for all or part of the E1 (E1A and/or E1B), E2, and/or E4 region, for example.
A further specific subject of the invention concerns a composition comprising two adenoviruses such as set forth hereinabove.
Another, equally preferred variant embodiment of the invention concerns a composition comprising the whole set of genetic elements required for constituting a retroviral particle, incorporated in a single recombinant adenovirus.
The special advantage of this embodiment is based notably on its simplified implementation due to the use of a single adenovirus.
It is understood that the compositions according to the invention can be adapted by those skilled in the art according to the structure of the retroviral vector, and particularly the defective retroviral genes therein. Thus, the vector used may be defective for one, two or all the retroviral genes gag, pol and env. Depending on the case, the composition and distribution of adenoviruses in the compositions of the invention may be readily adapted. However, a preferred embodiment is that in which the retroviral vector is defective for the whole set of genes gag, pol and env, as illustrated in the examples, and that adapted to producing recombinant lentiviruses.
In the compositions of the invention, the nucleic acid used coding for a retroviral protein gag, pol or env generally comprises a transcriptional promoter located 5xe2x80x2 of the coding region, and a transcriptional terminator located 3xe2x80x2 of the coding region. The promoters used may vary in their nature, origin and properties. The choice of promoter depends in fact on the desired use and on the transgene, in particular. Thus, the promoter may be constitutive or regulated, strong or weak, ubiquitous or tissue/cell-specific, or even specific of physiological or pathophysiological states (activity dependent on the state of cell differentiation or the step in the cell cycle). The promoter may be of eukaryotic, prokaryotic, viral, animal, plant, artificial or human, etc., origin. Specific examples of promoters are the promoters of the genes PGK, TK, GH, EF1-xcex1, APO, CMV, etc. or artificial promoters, such as those for p53. E2F or cAMP.
Preferably, the promoter controlling the expression of gag, pol and env is a strong constitutive promoter. Moreover, the promoter may also comprise an xe2x80x9cenhancerxe2x80x9d region to increase the efficiency of expression. The choice of the terminator region may also be effortlessly made by those skilled in the art, particularly among the terminators of the genes GH, SV40, EF1-xcex1, etc., widely described in the literature. Finally, the nucleic acids coding for gag and pol are often used in the form of a bicistronic unit, controlled by a single promoter and terminator, as is the case in the retroviral genome.
As noted hereinabove, the advantages of the present invention derive notably from the distribution of the retroviral genetic elements in the adenoviruses, and/or from the type of adenovirus used. In this respect, as underscored above, the adenoviruses used in the context of the present invention are primarily defective for all or part of the E1 region and the E4 region at least. As illustrated in the examples, together with an advantageous distribution of retroviral genetic elements, this organization of the adenoviral genome procures high safety and efficiency. Furthermore, the genomic structure of the recombinant adenoviruses used also allows the production of original and advantageous distributions of the retroviral genetic elements, such as notably the use of a single adenovirus or of a combination of two adenoviruses as illustrated hereinabove. The results show, in an especially advantageous manner, that the use of this type of adenovirus and/or of specific distributions of retroviral genetic elements according to the invention makes it possible to obtain high titers of infectious retroviral particles and long-term stability of the transgene (beyond 3 months). These entirely advantageous results are surprising in so far as the absence of the viral E4 region has been described as having a negative effect on the expression of sequences introduced into an adenoviral vector. Furthermore, the results presented show that, in vivo, the compositions according to the invention induce at least a 10- to 50-fold increase in the number of transduced tumor cells, as compared to administration of an adenovirus alone. These results thus confirm the advantageous and surprising properties of the present invention for transferring nucleic acids into cells.
The adenoviruses used to carry out the present invention are therefore advantageously defective for all or part of the E1 and E4 regions at least.
Advantageously these are so-called third generation defective recombinant adenoviruses, i.e. defective for all or part of the E1 and E4 regions, and possibly for the E3 region.
Specific variants of the invention comprise the use of adenoviruses harboring deletions affecting all or a functional part of the following regions:
E1, E4 and E3,
E1, E4 and E2,
E1, E4, E2 and E3,
the regions hereinabove as well as all or part of the genes encoding the adenovirus late functions (L1 to L5), or further still,
all the viral coding regions.
The genomic structure of adenoviruses has been largely described in the literature. In this respect, the genome of adenovirus Ad5 has been fully sequenced and is accessible from data bases (see notably GeneBank M73260). Likewise, parts or even all of other adenovirus genomes (Ad2, Ad7, Ad12, canine to adenovirus CAV-2, etc.) have also been sequenced. Furthermore, the construction of defective recombinant adenoviruses has also been described in the literature. Thus, applications WO 94/28152, WO 95/02697 and WO 96/22378, for example, describe different deletions in the E1 and E4 regions. Similarly, application WO 96/10088 describes vectors bearing a modification in the Iva2 gene, application WO 94/26914 describes animal adenoviruses, and application WO 95/29993 describes deletions in the adenoviral E2 region.
Advantageously, the recombinant adenovirus used in the context of the invention comprises a deletion in the E1 region of its genome affecting the E1a and E1b regions. A specific example is provided by deletions affecting nucleotides 454-3328, 382-3446 or 357-4020 (with reference to the Ad5 genome).
Furthermore, the deletion in the E4 region preferentially affects all the open reading frames, such as for example deletions 33466-35535 or 33093-35535, or only part of the E4 region (ORF6 or ORF3 for example), as described in applications WO95/02697 and WO96/22378, incorporated herein as reference.
As for adenoviruses further deleted for late functions (xe2x80x9cminimumxe2x80x9d vector) or for all coding regions (xe2x80x9cgutlessxe2x80x9d vector), their construction has been described for instance by Parks et al., PNAS (1996) 93: 13565 and Lieber et al., J. Virol. (1996) 70: 8944.
The retroviral genetic elements may be inserted at different sites in the recombinant adenoviral genome. They may be inserted in the E1, E3 or E4 region, by replacing the deleted sequences or in addition. They may also be inserted at any other site, apart from sequences required in cis for virus production (ITR sequences and packaging sequence).
Moreover, the recombinant adenoviruses may be of human or animal origin. As far as adenoviruses of human origin are concerned, those in group C may preferentially be cited, in particular the adenoviruses type 2 (Ad2), type 5 (Ad5); or adenoviruses type 7 (Ad7) or 12 (Ad12). Adenoviruses of animal origin are preferentially exemplified by the canine adenoviruses, and particularly all the strains of adenovirus CAV2 [Manhattan strain or A26/61 (ATCC VR-800), for example]. Other adenoviruses of animal origin are given notably in application WO94126914, incorporated herein as reference.
The recombinant adenoviruses are produced in packaging cells, i.e. a cell line that can complement in trans one or several of the deficient functions in the recombinant adenoviral genome. Among the packaging cells familiar to those skilled in the art, an example is cell line 293 in which part of the adenovirus genome has been integrated. More specifically, cell line 293 is a human embryonic kidney cell line containing the left extremity (approximately 11-12%) of the genome of adenovirus serotype 5 (Ad5), containing the left ITR, the packaging region, the E1 region, including E1a and E1b, the region encoding the pIX protein and part of the region encoding the plVa2 protein. This cell line is able to trans-complement recombinant adenoviruses defective for the E1 region, i.e. deleted of all or part of the E1 region, and produce viral stocks at high titers. This cell line can also produce, at permissive temperature (32xc2x0 C.), stocks of virus further comprising the temperature-sensitive E2 mutation. Other cell lines capable of complementing the E1 region have been described, based notably on human lung carcinoma cells A549 (WO94/28152) or on human retinoblasts [Hum.
Gen. Ther. (1996) 215]. Moreover, cell lines capable of trans-complementing several adenoviral functions have also been described. Specific examples comprise the cell lines complementing the E1 and E4 regions [Yeh et al., J. Virol. (1996) 70: 559-565; Cancer Gen. Ther. (1995) 2: 322; Krougliak et al., Hum. Gen. Ther. (1995) 6: 15751 and cell lines complementing the E1 and E2 regions (WO94/28152, WO95/02697, WO95/27071) or cell lines derived therefrom that can be used for producing minimum adenoviruses, especially since they also express site-specific recombinase activity involved in the construction of such viruses.
The recombinant adenoviruses are normally produced by introducing viral DNA into the packaging cells, followed by cell lysis after about 2 or 3 days (the adenoviral replication cycle being 24 to 36 hours). To carry out the method, the introduced viral DNA may be the complete recombinant viral genome, possibly constructed in bacteria (WO96/25506) or yeast (WO95/03400), transfected into the cells. It may also be a recombinant virus used to infect the packaging cells. The viral DNA may also be introduced in the form of fragments each bearing a part of the recombinant viral genome and a region of homology which, after introduction into the packaging cell, allows reconstitution of the recombinant viral genome by means of homologous recombination between the different fragments.
Following cell lysis, the recombinant viral particles can be isolated by any known method such as cesium chloride gradient centrifugation or chromatography. An alternative method was notably described in application FR 9608164, incorporated herein as reference.
The compositions according to the invention may comprise variable amounts of recombinant adenovirus, easily adaptable by those skilled in the art according to the desired application (in vitro, ex vivo or in vivo, for instance).
Generally, the compositions comprise approximately 105 to 1015 v.p. of each recombinant adenovirus, preferably 107 to 1012 v.p.
The term v.p. corresponds to the number of viral particles present in the compositions.
Furthermore, the compositions of the invention may also be in different forms, such as solutions, gels, powder, etc. They are generally solutions, preferably sterile, such as for instance saline solutions (monosodium phosphate, disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of these salts), isotonic, or dry compositions, particularly lyophilizates which, by addition of sterile water or ohysiological serum, as the case may be, form reconstituted solutions. Other excipients may be used such as for example a hydrogel. Such a hydrogel may be prepared from any biocompatible, non-cytotoxic (homo or hetero) polymer. Such polymers are described in application WO93/08845 for example.
In addition, the compositions of the invention may be packaged in any suitable type of device, such as a bottle, tube, ampoule, bag, syringe, balloon, etc. Further, in compositions of the invention comprising at least two types of recombinant adenovirus, the latter may be packaged either as a mixture or individually.
As indicated hereinabove, the compositions of the invention may be used in vitro, ex vivo or in vivo.
For in vitro or ex vivo use, the cells may simply be incubated, in any suitable device (plate, dish, bag, etc.) in the presence of a composition such as described hereinabove. As shown in FIG. 1 for a composition comprising two types of recombinant adenovirus, incubation of the cells causes the cells to be infected by the two adenoviruses, after which the coinfected cell can produce infectious retroviral particles. For this type of application, the compositions may be used at multiplicities of infection (MOI) of between 10 and 5000 v.p. per cell, for instance, preferably between 100 and 2000, as shown in particular in the examples.
In this respect, the invention equally concerns a method for producing retroviral particles in vitro comprising incubating cells in the presence of a composition such as described hereinabove, possibly followed by recovery and/or purification of the retroviruses produced. The cells that can be used in this method may be any type of cell permissive to adenovirus. These may be primary cultures or cell lines, particularly mammalian, and particularly of human origin. The use of cells of human origin (embryonic kidney cells, A549, retinoblasts, HeLa, KB, etc.) is especially advantageous because it gives the retroviral particles heightened resistance to the complement system. As shown in the examples, the method of the invention makes it possible to obtain high retroviral titers, without the use of packaging cell lines.
For it vivo use, the compositions of the invention may be formulated in is view of administration by the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratumoral, etc. route.
In this respect, the invention further concerns a method for transferring a nucleic acid in vivo comprising administering a composition such as described hereinabove. The invention may be used for instance in animals, to establish pathological models, or for the study of gene regulation, and also in humans, in labeling or bioavailability studies, or for medical purposes. The invention is also directed to the use of the compositions described hereinabove for producting infectious retroviral particles in vivo.
According to the nucleic acid of interest (transgene) inserted in the retroviral vector, the present invention may be used in a large number of applications. Thus, among the products of interest in the context of the present invention, more specific examples include enzymes, blood products, hormones such as growth hormone, cytokines, lymphokines:intedeukins, interferons, TNF, etc. (French patent 92 03120), growth factors, for example angiogenic factors such as VEGF or FGF, neurotransmitters or their precursors or synthesis enzymes, trophic factors, particularly neurotrophic factors for the treatment of neurodegenerative diseases or nervous system trauma, or macular degeneration :BDNF, CNTF, NGF, IGF, GMF aFGF, NT3, NT5, HARP/pleiotrophin, or bone growth factors, hematopoietic factors, etc., dystrophin or minidystrophin (French patent 91 11947), genes encoding coagulation factors:factors VII, VIII, IX, suicide genes (thymidine kinase, cytosine deaminase), proteins involved in the cell cycle such as p21, or other kinasedependent inhibitor proteins, Rb, Gas-1, Gas-6, Gas-3, Gad 45, Gad 153, cyclins A, B, D, or further still the GAX protein inhibiting smooth muscle cell proliferation (treatment of restenosis), apoptosis-inducing proteins or other tumor suppressors such as p53, Bax, BcIX-s, Bad or any other antagonist of Bcl2 and BcIX-1, genes for hemoglobin or other transport proteins, genes for proteins involved in lipid metabolism, of the apolipoprotein type chosen from among apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-II, D, E, F, G, H, J and apo(a), metabolic enzymes such as for instance lipoprotein lipase, hepatic lipase, cholesterol lecithin acyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidyl acid phosphatase, or still lipid transport proteins such as cholesterol ester transport protein and phospholipids transport protein, HDL binding protein or further still a receptor chosen from the LDL receptors, chylomicron-remnant receptors and scavenger receptors, etc.
Among the products of interest it is important to point out antibodies, antibody single chain variable fragments (ScFv) or any other antibody fragment with recognition capabilities for its use in immunotherapy, for example in the treatment of infectious diseases, tumors (anti-RAS, anti-p53 or anti-GAP antibodies), autoimmune diseases such as multiple sclerosis (anti-idiotype antibody).
Non-limiting examples of other proteins of interest are the soluble receptors, such as for instance the soluble CD4 receptor or the soluble TNF receptor for anti-HIV therapy, the soluble acetylcholine receptor for the treatment of myasthenia gravis; peptides which are enzyme inhibitors or substrates, or peptides which agonize or antagonize receptors or adhesion proteins such as for instance for the treatment of asthma, thrombosis and restenosis:synthetic, chimeric or truncated proteins. Among the hormones of primary interest one may cite insulin in the case of diabetes, growth hormone and calcitonin.
The nucleic acid may also be a gene or an antisense sequence, whose expression in the target cell enables control of gene expression or transcription of cellular mRNA. Such sequences may, for instance, be transcribed in the target cell to RNA complementary to cellular mRNA, thereby lo blocking its translation to protein, according to the method described in European patent 140 308. Therapeutic genes equally comprise sequences coding for ribozymes, able to selectively destroy target RNAs (European patent 321 201).
The nucleic acid may also comprise one or several genes coding for an antigenic peptide, that can elicit an immune response in humans or animals. In this particular embodiment, the invention therefore allows the production either of vaccines, or of immunotherapy treatments for use in humans or animals, notably against microorganisms, viruses or cancers. In particular these may be antigenic peptides specific of Epstein Barr virus, HIV virus, hepatitis B virus (European patent 185 573), pseudo-rabies virus, xe2x80x9csyncitia forming virusxe2x80x9d, other viruses or yet tumor-specific antigens such as MAGE proteins (European patent 259 212).
The nucleic acid may also code for a product toxic to cells. particularly a conditional toxicity (eg., thymidine kinase, cytosine deaminase, etc.).
Other genes of interest have notably been described by Mc Kusick, V. A. Mendelian [Inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked phenotypes, Eighth edition. John Hopkins University Press (1988)], and by Standbury, J. B. et al. [The metabolic basis of inherited disease, Fifth edition. McGraw-Hill (1983)]. The genes of interest comprise the proteins involved in the metabolism of amino acids, lipids and other components of the cell.
Finally, the nucleic acid may comprise several coding regions, possibly separated by an IRES, allowing the production of several products of interest.
Furthermore, the transgene generally comprises a transcriptional promoter (located 5xe2x80x2) and a transcriptional terminator (located 3xe2x80x2), which may be chosen by those skilled in the art, as described hereinabove. In addition, the transgene may be present in the retroviral vector in the same or the opposite orientation to the direction of LTR transcription. Finally, in a particular embodiment, the adenoviral genomes of the invention may also be delivered to cells in the form of plasmids, together with adenoviral complementation functions.
Other advantages and applications of the present invention will become more apparent from the following examples, which are given for purposes of illustration and not by way of limitation.